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Acute effects of aflatoxin B1 on tRNA methylase function
Pergamon Press
Life Sciences Vol . 17, pp . 519-522 Printed in the U.S .A .
ACUTE EFFECTS OF AFLATOXIN B 1 William F .
Busby,
Jr .,
ON tRNA METHYLASE
Pamela M .
Department of Nutrition Massachusetts Cambridge,
FUNCTION
Hurley and Gerald
N . Wogan
and Food Science
Institute of Technology Massachusetts
02139
(Received in final form July 7, 1975)
Su mmary Transfer RNA methylase activity and capacity were measured in relation to the acute effects of aflatoxin Dose-independent methylase activity B 1 on rat liver . was elevated approx . 40~ within 3 days after dosing, and gradually declined towards control values over a 3-week period . Transfer RNA methylase capacity, in contrast, exhibited a linear dose-response relationship with values elevated as much as 100 over control levels . Transfer RNA methylases are a complex group of species-, organ- and site specific enzymes which catalyze the methylation of pre-formed tRNA (1) . Increased levels of tRNA methylase activity (a rate function assayed with excess heterologous tRNA and limiting enzyme) and capacity (a measurement of the maximum incorporation of methyl groups with excess enzyme and limiting heterologous tRNA) have been consistently observed in a wide range of tumors and transformed cells (2-4) . Recent investigations (5) have shown that one aspect of elevated tRNA methylase activity and capacity noted during the initial stages of aflatoxin B 1 (AFB1)induced hepatocarcinogenesis may be related to the acute toxic effects of the carcinogen . The data presented here represented a further attempt at describing the effects of a single acute dose of AF~ 1 on tRNA methylase function with the objective of investigating alterations in overall tRNA methylase function as an indicator of cell toxicity . Methods Weanling male Fischer rats (Charles River Breeding Laboratories, Wilmin ton, Mass .) were randomized and given access to agar gel diet ~6) and water ad Zibitum . Animals (100-150 g) were injected i .p . with AFB 1 (0 .375, 0 .75 or 1 .5 mg/kg corresponding to 1/8, 1/4 and 1/2 LD SO dose, respectively) in 50 ul dimethyl sulfoxide (DMSO) . Control animals received 50 ul DMSO only . A cytosol enzyme fraction was prepared from livers of decapitated animals with a procedure modified from Kerr (7) . Liver was minced with a tissue press, homogenized with 4 vol . medium A (0 .4 M sucrose ; 10 mM tris-HCI, pH 7 .5 ; 6 mM MgC1 2 ; 5 mM 2-mercaptoethanol), and centrifuged for 10 min . a t 15,000 x g and 519
520
Aflataxin and t1aiA Methylase Function
Vol . 17, No . 4
105,000 x g for 90 min . The cytosol supernatant was diluted with 0 .9 ml medium A and 0 .1 ml 100 mM 2-mercaptoethanol per ml preProtein was determined by the Lowry (8) paration before use . procedure . Methylase assays were run in a final volume of 0 .25 ml at 37° using a modification of Kerr (7) . Activity assays contained : tris-HC1 (pH 8 .2), 12 .5 ~Imoles ; MgC1 2 , 1 .25 ymoles ; 2-mercaptoethanol, 1 .25 umoles ; [ 1 C]-S-adenosyl-L-methionine (58 mCi/mmole ; New England Nuclear Corp ., Boston, Mass .), 2 .5 nmoles ; rs . coli B tRNA (Schwarz-Mann, Orangeburg, N .J .), 125 ug ; and approx . 0 .75 mg enzyme protein . Capacity assays were run with 6 .7 ug E . aoti B tRNA and approx . 1 .3 mg enzyme protein . Blank assays were run without tRNA addition . Aliquots (0 .05 ml) were removed and placed on glass fiber discs (Whatman GF/A, 2 .4 cm dia .) at 15, 30 and 45 min . for activity assays and 60, 75 and 90 min . for capacity assays . The discs were placed in lOX ice-cold trichloroacetic acid for 60 min ., drained and stored in diethyl ether until washed (9) . Capacity values were determined by averaging the plateau levels at 60, 75 and 90 min . ; activity values were calculated at the 30 min . intercept by linear regression analysis . Methylase activity assays were linear with protein levels at least twice the assay protein concentration . Capacity assays exhibited na additional increase in tRNA methyl group incorporation at two times the normal enzyme level . Results Animals were dosed with 1/8, 1/4 and 1/2 LD so amounts of AFB 1 and tRNA methylase activities monitored over a 3-week period (FIG . 1) . Acute liver damage (lipid accumulation) was grossly apparent c ~E O e
70 BO
ô
50
E
40
n 0 °
c
"Ml S
V ~Û i E n
30 2D 10 0
C 0 DAYS
C 1/8 I/4 1/2
0 ~ 1~9 I/4 1/P
3 DAYS
7 DAYS
FIG .
C I/8 I/4 I I2 14 PAYS
C
I/8 I/4
PI DAYS
1
Transfer RNA methylase activity of control (C) and AFB 1 treated rats at various times after i .p . injection of 1/8 LDso (1/8), 1/4 LD so (1/4) and 1/2 LD s4 (1/2) AFB 1 doses . Separate enzyme preparations were assayed in duplicate from 3 animals at each dose and time interval . Results are expressed as S .E .M .
Vol . 17, No . 4
Aflatozin anS tRNA Methylase Fuaction
521
in all groups after 3 days, with relatively little damage noted in With the exception of the 1/2 LDso-dosed the 1/8 LD so animals . animals, in which diminished liver size and ascites was present, gross liver appearance gradually attained normal limits in the other groups . Maximal elevation of activity (approx . 40%) occurred at least as early as 3 days post-dosing and gradually A sufficient number of 1/2 declined to approx . 15% after 3 weeks . LD so -dosed animals did not survive for a 3-week methylase activity determination . These data were subjected to a two-way analysis of variance . No significant differences in methylase activity (p > 0 .1) were noted among the 3 AFB 1 -dosed groups during the first 14 days of the experiment, although the activities of the treated animal groups were significantly higher (p <0 .02) than control animal activities . Thus, increased tRNA methylase activity noted in treated animals was independent of the AFB, dosages used in this experiment . A second experiment demonstrating the effect of AFB 1 -dosing on tRNA methylase capacity 7 days post-treatment is depicted in FIG . 2 . Again gross evidence of hepatic lipid accumulation was apparent . Unlike methylase activity, methylase capacity exhibited a linear dose-response relationship, with values for 1/2 LDsotreated animal methylases being approx . 100% higher than control levels .
aa
= 0.4 t
ô
sU a
42
4~
0
0
o~a
4a
mp AFBy lp/kq
FIG .
2
Dose-response relationship of tRNA methylase capacity 7 days post dosing with 1/8, 1/4 and 1/2 LD so AFB 1 doses . Separate enzyme preparations were assayed in duplicate from 3 animals at each dose level . Results are expressed as S .E .M .
522
Aflatoxin and tFàiA Methylase Function
Vol . 17, No . 4
Discussion These data describe the elevation of tRNA methylase activity and capacity in rat liver in response to acute doses of AFB 1 . Although no previous studies of tRNA methylase capacity have been reported under acute conditions, increased tRNA methylase activity has resulted from the effects of dimethylnitrosamine on rat kidney (10), ethionine on rat liver and dimethylaminoazobenzene on hamster liver (11) . These elevations in tRNA methylase function may be due more to the acute toxic effects of the carcinogen rather than to its cell transformation potential . This hypothesis is strengthened by the fact that ethionine is not hepatocarcinogenic in the hamster (11) . The mechanisms for the expression of these altered methylase functions is unclear . Either the presence of activators, such as certain polyamines (12), or the relative absence of inhibitors or com etitors such as the glycine methyltransferase system of Kerr It is of (13~ would result in elevated methylase function . particular interest that decreased glycine methyltransferase activity has been implicated in the increased tRNA methylase function in tumors (14), but not in the acute response of rat Experiments comparing methylated liver to ethionine feeding (11) . reparations base patterns of heterologous tRNA using methylase from both the acute (toxic) and chronic (neoplastic~ state might prove enlightening . Ackn owledgements Financial support was provided by Grant No . 5 PO1 from NIEHS and Contract No . NIH NO1-CP-43265 from NCI .
ES 00597
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 .
S . J . KERR and E . BOREK . Adv . ~Enz m_ . ,~¢ :1-27 (1972) . ~ . C ancer Res . :163-190 (1972) . E . BOREK and S . J . KERR . M . J . KLAGSBRUN . J . Biol . Chem . ~ :7~-7 1 (1972) . A . E . PEGG and A . Ff . ~WKS . ~ochem . J . ~ :229-238 (1974) . and G . N . WOGAN . W . F . BUSBY, S . PAGLIALUNGA, P . M .~NEWBERN Cancer Res . (Submitted for publication) . Cancer _Res . 28 :770-781 P . M . NEWBERNE and G . N . WOGAN . (1968) . Biochemistr .~ :690-695 (1970) . S . J . KERR . 0 GH, A . L . FARR and R . J . RANDALL . 0 . H . LOWRY, J . Biol . Chem .~j,~ :265-275 (1951) . 17 . 1rßUSg~and P . HELE . Biochim . Biophys . Act a 224 :413-422 (1970) . Biochim . Biophys . Act a 281 : B . W . STEWART and A . E . PEGG . 416-424 (1972) . Cancer _Res . 33 :1747-1753 R . L . HANCOCK and P . I . FORRESTER . (1973) . P . LEBOY . Bioch e~mistr~ 9 :1577-1584 (1970) . J . Bio1 . Chém . 247 :4248-4252 (1972) . S . J . KERR . S . J . KERR . isroc .Tla t~ad~ci . USA 68 :406-410 (1971) .
Life Sciences Vol . 17, pp . 519-522 Printed in the U.S .A .
ACUTE EFFECTS OF AFLATOXIN B 1 William F .
Busby,
Jr .,
ON tRNA METHYLASE
Pamela M .
Department of Nutrition Massachusetts Cambridge,
FUNCTION
Hurley and Gerald
N . Wogan
and Food Science
Institute of Technology Massachusetts
02139
(Received in final form July 7, 1975)
Su mmary Transfer RNA methylase activity and capacity were measured in relation to the acute effects of aflatoxin Dose-independent methylase activity B 1 on rat liver . was elevated approx . 40~ within 3 days after dosing, and gradually declined towards control values over a 3-week period . Transfer RNA methylase capacity, in contrast, exhibited a linear dose-response relationship with values elevated as much as 100 over control levels . Transfer RNA methylases are a complex group of species-, organ- and site specific enzymes which catalyze the methylation of pre-formed tRNA (1) . Increased levels of tRNA methylase activity (a rate function assayed with excess heterologous tRNA and limiting enzyme) and capacity (a measurement of the maximum incorporation of methyl groups with excess enzyme and limiting heterologous tRNA) have been consistently observed in a wide range of tumors and transformed cells (2-4) . Recent investigations (5) have shown that one aspect of elevated tRNA methylase activity and capacity noted during the initial stages of aflatoxin B 1 (AFB1)induced hepatocarcinogenesis may be related to the acute toxic effects of the carcinogen . The data presented here represented a further attempt at describing the effects of a single acute dose of AF~ 1 on tRNA methylase function with the objective of investigating alterations in overall tRNA methylase function as an indicator of cell toxicity . Methods Weanling male Fischer rats (Charles River Breeding Laboratories, Wilmin ton, Mass .) were randomized and given access to agar gel diet ~6) and water ad Zibitum . Animals (100-150 g) were injected i .p . with AFB 1 (0 .375, 0 .75 or 1 .5 mg/kg corresponding to 1/8, 1/4 and 1/2 LD SO dose, respectively) in 50 ul dimethyl sulfoxide (DMSO) . Control animals received 50 ul DMSO only . A cytosol enzyme fraction was prepared from livers of decapitated animals with a procedure modified from Kerr (7) . Liver was minced with a tissue press, homogenized with 4 vol . medium A (0 .4 M sucrose ; 10 mM tris-HCI, pH 7 .5 ; 6 mM MgC1 2 ; 5 mM 2-mercaptoethanol), and centrifuged for 10 min . a t 15,000 x g and 519
520
Aflataxin and t1aiA Methylase Function
Vol . 17, No . 4
105,000 x g for 90 min . The cytosol supernatant was diluted with 0 .9 ml medium A and 0 .1 ml 100 mM 2-mercaptoethanol per ml preProtein was determined by the Lowry (8) paration before use . procedure . Methylase assays were run in a final volume of 0 .25 ml at 37° using a modification of Kerr (7) . Activity assays contained : tris-HC1 (pH 8 .2), 12 .5 ~Imoles ; MgC1 2 , 1 .25 ymoles ; 2-mercaptoethanol, 1 .25 umoles ; [ 1 C]-S-adenosyl-L-methionine (58 mCi/mmole ; New England Nuclear Corp ., Boston, Mass .), 2 .5 nmoles ; rs . coli B tRNA (Schwarz-Mann, Orangeburg, N .J .), 125 ug ; and approx . 0 .75 mg enzyme protein . Capacity assays were run with 6 .7 ug E . aoti B tRNA and approx . 1 .3 mg enzyme protein . Blank assays were run without tRNA addition . Aliquots (0 .05 ml) were removed and placed on glass fiber discs (Whatman GF/A, 2 .4 cm dia .) at 15, 30 and 45 min . for activity assays and 60, 75 and 90 min . for capacity assays . The discs were placed in lOX ice-cold trichloroacetic acid for 60 min ., drained and stored in diethyl ether until washed (9) . Capacity values were determined by averaging the plateau levels at 60, 75 and 90 min . ; activity values were calculated at the 30 min . intercept by linear regression analysis . Methylase activity assays were linear with protein levels at least twice the assay protein concentration . Capacity assays exhibited na additional increase in tRNA methyl group incorporation at two times the normal enzyme level . Results Animals were dosed with 1/8, 1/4 and 1/2 LD so amounts of AFB 1 and tRNA methylase activities monitored over a 3-week period (FIG . 1) . Acute liver damage (lipid accumulation) was grossly apparent c ~E O e
70 BO
ô
50
E
40
n 0 °
c
"Ml S
V ~Û i E n
30 2D 10 0
C 0 DAYS
C 1/8 I/4 1/2
0 ~ 1~9 I/4 1/P
3 DAYS
7 DAYS
FIG .
C I/8 I/4 I I2 14 PAYS
C
I/8 I/4
PI DAYS
1
Transfer RNA methylase activity of control (C) and AFB 1 treated rats at various times after i .p . injection of 1/8 LDso (1/8), 1/4 LD so (1/4) and 1/2 LD s4 (1/2) AFB 1 doses . Separate enzyme preparations were assayed in duplicate from 3 animals at each dose and time interval . Results are expressed as S .E .M .
Vol . 17, No . 4
Aflatozin anS tRNA Methylase Fuaction
521
in all groups after 3 days, with relatively little damage noted in With the exception of the 1/2 LDso-dosed the 1/8 LD so animals . animals, in which diminished liver size and ascites was present, gross liver appearance gradually attained normal limits in the other groups . Maximal elevation of activity (approx . 40%) occurred at least as early as 3 days post-dosing and gradually A sufficient number of 1/2 declined to approx . 15% after 3 weeks . LD so -dosed animals did not survive for a 3-week methylase activity determination . These data were subjected to a two-way analysis of variance . No significant differences in methylase activity (p > 0 .1) were noted among the 3 AFB 1 -dosed groups during the first 14 days of the experiment, although the activities of the treated animal groups were significantly higher (p <0 .02) than control animal activities . Thus, increased tRNA methylase activity noted in treated animals was independent of the AFB, dosages used in this experiment . A second experiment demonstrating the effect of AFB 1 -dosing on tRNA methylase capacity 7 days post-treatment is depicted in FIG . 2 . Again gross evidence of hepatic lipid accumulation was apparent . Unlike methylase activity, methylase capacity exhibited a linear dose-response relationship, with values for 1/2 LDsotreated animal methylases being approx . 100% higher than control levels .
aa
= 0.4 t
ô
sU a
42
4~
0
0
o~a
4a
mp AFBy lp/kq
FIG .
2
Dose-response relationship of tRNA methylase capacity 7 days post dosing with 1/8, 1/4 and 1/2 LD so AFB 1 doses . Separate enzyme preparations were assayed in duplicate from 3 animals at each dose level . Results are expressed as S .E .M .
522
Aflatoxin and tFàiA Methylase Function
Vol . 17, No . 4
Discussion These data describe the elevation of tRNA methylase activity and capacity in rat liver in response to acute doses of AFB 1 . Although no previous studies of tRNA methylase capacity have been reported under acute conditions, increased tRNA methylase activity has resulted from the effects of dimethylnitrosamine on rat kidney (10), ethionine on rat liver and dimethylaminoazobenzene on hamster liver (11) . These elevations in tRNA methylase function may be due more to the acute toxic effects of the carcinogen rather than to its cell transformation potential . This hypothesis is strengthened by the fact that ethionine is not hepatocarcinogenic in the hamster (11) . The mechanisms for the expression of these altered methylase functions is unclear . Either the presence of activators, such as certain polyamines (12), or the relative absence of inhibitors or com etitors such as the glycine methyltransferase system of Kerr It is of (13~ would result in elevated methylase function . particular interest that decreased glycine methyltransferase activity has been implicated in the increased tRNA methylase function in tumors (14), but not in the acute response of rat Experiments comparing methylated liver to ethionine feeding (11) . reparations base patterns of heterologous tRNA using methylase from both the acute (toxic) and chronic (neoplastic~ state might prove enlightening . Ackn owledgements Financial support was provided by Grant No . 5 PO1 from NIEHS and Contract No . NIH NO1-CP-43265 from NCI .
ES 00597
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 .
S . J . KERR and E . BOREK . Adv . ~Enz m_ . ,~¢ :1-27 (1972) . ~ . C ancer Res . :163-190 (1972) . E . BOREK and S . J . KERR . M . J . KLAGSBRUN . J . Biol . Chem . ~ :7~-7 1 (1972) . A . E . PEGG and A . Ff . ~WKS . ~ochem . J . ~ :229-238 (1974) . and G . N . WOGAN . W . F . BUSBY, S . PAGLIALUNGA, P . M .~NEWBERN Cancer Res . (Submitted for publication) . Cancer _Res . 28 :770-781 P . M . NEWBERNE and G . N . WOGAN . (1968) . Biochemistr .~ :690-695 (1970) . S . J . KERR . 0 GH, A . L . FARR and R . J . RANDALL . 0 . H . LOWRY, J . Biol . Chem .~j,~ :265-275 (1951) . 17 . 1rßUSg~and P . HELE . Biochim . Biophys . Act a 224 :413-422 (1970) . Biochim . Biophys . Act a 281 : B . W . STEWART and A . E . PEGG . 416-424 (1972) . Cancer _Res . 33 :1747-1753 R . L . HANCOCK and P . I . FORRESTER . (1973) . P . LEBOY . Bioch e~mistr~ 9 :1577-1584 (1970) . J . Bio1 . Chém . 247 :4248-4252 (1972) . S . J . KERR . S . J . KERR . isroc .Tla t~ad~ci . USA 68 :406-410 (1971) .